Since Tiselius pioneered electrophoretic separation of human serum albumin, xcex1-, xcex2- and xcex3- globulin in 1937, electrophoresis of biological molecules has been critical to biomedical research (1). Electrophoretic analysis has become more sophisticated, specialized and useful as new types of electrophoresis are developed (2,3). McDonell et al. and Southern offered detailed descriptions of standard agarose gel electrophoresis and its use for DNA analysis (4,5). Pulsed-field agarose gel electrophoresis is an alternative for separation of very large DNA fragments up to 2000 kb (6). Another important application is polyacrylamide gel electrophoresis for separation of small DNA segments, such as dideoxy sequencing analysis (7,8) and SSCP analysis (9-11).
Electrophoresis of nucleic acids in agarose and polyacrylamide gels is generally performed with TAE or TBE buffers. These buffers perform well in many applications, but certain limitations exist. A key limitation is buffering capacity which determines the working concentration and, in turn, determines the rate at which electrophoresis can occur without distortions due to heating. Limiting buffer capacity may require a change of buffer when long electrophoresis times are required, e.g., in mutation scanning using restriction endonuclease fingerprinting or SSCP. TAE buffer cannot be used for sequencing gels because of its low buffering capacity and TAE has a relatively low solubility, such that the maximal stock solution is 20X (2,3). Typically, laboratories that perform sequencing or SSCP-type mutation scanning prepare large volumes of stock solution.
This invention provides pK-matched buffers comprising a mixture of a weak acid and a weak base which have pKa values at 25xc2x0 C. within about 0.3 units of one another. The buffers are useful as running buffers for gel electrophoresis of nucleic acids or polypeptides. These pK-matched buffers have the following advantages relative to standard TBE and TAE electrophoresis buffers: 1) high resolution, 2) high electrophoretic stability, 3) low working concentration, and 4) a wide range of pH values for selection.
The invention includes an improved gel electrophoresis method of separating nucleic acids or polypeptides. The improvement comprises running the electrophoresis in a pK-matched buffer of the invention.